Inbred corn line KWU7104

ABSTRACT

This invention is in the field of corn breeding, specifically relating to a dent corn inbred designated as KWU7104. Inbred KWU7104 is a Lancaster-Mo17 family based inbred that is specifically bred by means of a pedigree selection method for the north central United States. As one of the parents, Inbred KWU7104 contributes superior test weight to the F1 hybrid. The specific density of the grain is referred to as test weight and is an important agronomic trait in the corn industry.

FIELD OF THE INVENTION

This invention is in the field of corn breeding, specifically relating to a dent corn inbred designated as KWU7104. Inbred KWU7104 is a Lancaster-Mo17 family based inbred that is specifically bred by means of a pedigree selection method for the north central United States. As one of the parents, Inbred KWU7104 contributes superior test weight to the F1 hybrid. Specific density of the grain is referred to as test weight and is an important agronomic trait in the corn industry. When appropriate industry standards that use a Lancaster-Mo17 family based inbred as one of the parents are compared to an F1 with KWU7104 as one of the parent lines, the superior test weight resulting from the genetics of KWU7104 is significant.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive corn inbred line, designated KWU7104. There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals.

The goal of plant breeding in corn is to develop inbred parent lines that contribute various desirable traits to the hybrids in which they are used. These traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, stalk strength, root strength, ear retention, maturity and plant and ear height, are important.

Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F.sub 1 hybrid cultivar, pureline cultivar, etc.) For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.

Field crops can be bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.

Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. A cross between two homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants each heterozygous at a number of loci will produce a population of hybrid plants that differ genetically and will not be uniform.

Corn plants (Zea mays L.) can be bred by both self-pollination and cross-pollination techniques. Corn has separate male and female flowers on the same plant, located on the tassel and the ear respectively. Natural pollination occurs in corn when the wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears.

The development of corn hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. The goal of plant breeding is to develop new, unique and superior corn inbred lines and hybrids. In pedigree selection breeding, the breeder combines the genetic backgrounds of two or more inbred lines or various broad-based sources into breeding pools from which the new inbred lines are developed by selfing and selection of the desired phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s).

Pedigree selection breeding starts with the crossing of two genotypes, each of which may have one or more desirable traits or more desirable characteristics that are lacking in the other or which complement the other. If the two original parents do not provide all of the desired characteristics, other sources can be included in the breeding population. In the pedigree selection method, superior plants are selfed and selected in successive generations. In the succeeding generations, the heterozygous condition gives way to the homozygous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more generations of selfing and selection are practiced: F1; F2; F3; F4; F5, etc. These selfing generations are sometimes designated as S0, S1, S2, etc with S0 being an equivalent to F1 while S2 is an equivalent to F3, etc.

Descriptions of breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., Allard, R. W. “Principles of Plant Breeding” John Wiley and Son, pp. 115-161, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr, 1987).

A single cross corn hybrid is the cross of two inbred parent lines, each of which has a genotype which compliments the genotype of the other. The hybrid progeny of the first generation is designated F1. In the development of hybrids, only the F1 hybrid plants are sought. Preferred F1 hybrids are more vigorous than their inbred parents. This hybrid vigor, or heterosis, can be manifested in many polygenic traits, including increased vegetative growth and increased yield.

The development of a hybrid corn variety involves three steps: (1) the selection of plants from various germplasm pools; (2) the selfing of the selected plants for several generations to produce a series of inbred lines, which, although different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with unrelated inbred lines to produce the hybrid progeny (F1). During the inbreeding process in corn, the vigor of the lines decreases. Vigor is restored when two unrelated inbred lines are crossed to produce the hybrid progeny (F1). An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between any two inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be produced indefinitely as long as the homogeniety of the inbred parents is maintained.

A single cross hybrid is produced when two inbred lines are crossed to produce the F1 progeny. Much of the hybrid vigor exhibited by F1 hybrids is lost in the next generation (F2). Consequently, seed from hybrid varieties is not used for planting stock.

Corn is an important and valuable field crop. Thus, a continuing goal of plant breeders is to develop consistent performing, high-yielding corn hybrids that are agronomically sound based on stable inbred lines. The reasons for this goal are obvious: to maximize the amount of grain produced with the inputs used and to minimize susceptibility of the crop to environmental stresses. To accomplish this goal, the corn breeder must select and develop superior inbred parental lines for producing hybrids. This requires identification and selection of genetically unique individuals which in a segregating population occur as a result of a combination of crossover events plus the independent assortment of specific combinations of alleles at many gene loci which results in specific genotypes. Based on the number of segregating genes, the frequency of occurrence of an individual with a specific genotype is less than 1 in 10,000. Thus, even if an entire genotype of the parents has been characterized and the desired genotype is know, only a few, if any, individuals having the desired genotype may be found in a large F2 or S1 population. Typically, however, the genotype of neither the parents nor the desired genotype is known in any detail. An agronomically acceptable F1 hybrid will come from a cross between two superior inbred parental lines. There is no assurance that either of these parental lines will produce a superior hybrid when crossed with a different inbred parent line. Thus, the selection or combination of the two parental inbreds provides a unique hybrid that demonstrates characteristics and performance levels that differ from that obtained when either of the parents is crossed with a different inbred parent line.

Once the superior combination of two parental lines is determined by the testing and selection of the F1 hybrid, that F1 hybrid and the performance traits and characteristics of the hybrid can be indefinitely reproduced so long as the parental inbreds are maintained in their homozygosity and the quality and production procedures are accomplished to the purity standards determined by the seed industry regulations. SUMMARY OF THE INVENTION

This invention provides for a novel inbred corn line designated as KWU7104. This invention thus relates to the seeds of inbred corn line KWU7104. The invention also includes the corn plants produced by the seed of KWU7104 and other plants resulting from all or part of the genetics of KWU7104 and other resulting hybrids in which KWU7104 is one of the parents. In addition, this invention provides for a corn plant having the physiological and morphological characteristics of inbred KWU7104.

This invention also provides for the tissue cultures of regenerable cells of a plant derived directly from inbred KWU7104 especially where the tissue regenerates into plants having all or essentially all of the important morphological and physiological characteristics of inbred KWU7104. The plants regenerated from the tissue culture cells derived from inbred KWU7104 are also a part of this invention.

Inbred seed or hybrid seed produced utilizing the genetic contributions of a plant or plants derived from inbred KWU7104 are expressly included in this invention.

The inbred corn plant of the invention may further comprise, or have, a cytoplasmic factor, or other factor, that is capable of conferring male sterility. So, the invention further comprises a male sterile form of the inbred. Parts of the corn plant of the present invention are also provided, such as e.g., pollen obtained from an inbred plant and an ovule of the inbred plant.

Another objective of the invention is to provide methods for producing other inbred corn plants derived from inbred KWU7104. Inbred corn lines derived by the use of those methods are also part of the invention.

The invention also relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic corn plant produced by that method.

In another aspect, the present invention provides for single gene converted plants of KWU7104. The single transferred gene may preferably be a dominant or a recessive allele. Preferably, the single transferred gene will confer such trait as male sterility, herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, enhanced nutritional quality and industrial usage. The single gene may be a naturally occurring maize gene or a transgene introduced through genetic engineering techniques.

The invention further provides for developing a corn plant in a corn plant breeding program using plant breeding techniques including recurrent selection, backcrossing, pedigree breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection and transformation, and haploid induction and dihaploid formation. Seed, corn plant, and parties thereof produced by such breeding methods are also part of the invention.

DEFINITIONS

In the description and tables that follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

-   -   Allele. The allele is any of one or more alternative form of a         gene, all of which alleles relates to one trait or         characteristic. In a diploid cell or organism, the two alleles         of a given gene occupy corresponding loci on a pair of         homologous chromosomes.     -   Backcrossing. Backcrossing is a process in which a breeder         repeatedly crosses hybrid progeny back to one of the parents,         for example, a first generation hybrid F₁ with one of the         parental genotype of the F₁ hybrid.     -   Essentially all the physiological and morphological         characteristics. A plant having essentially all the         physiological and morphological characteristics means a plant         having the physiological and morphological characteristics,         except for the characteristics derived from the converted gene.     -   Regeneration. Regeneration refers to the development of a plant         from tissue culture.     -   Single gene converted. Single gene converted or conversion plant         refers to plants which are developed by a plant breeding         technique called backcrossing wherein essentially all of the         desired morphological and physiological characteristics of an         inbred are recovered in addition to the single gene transferred         into the inbred via the backcrossing technique or via genetic         engineering.     -   Predicted RM. This trait for a hybrid, predicted relative         maturity (RM), is based on the harvest moisture of the grain.         The relative maturity rating is based on a known set of checks         and utilizes conventional maturity such as the Comparative         Relative Maturity Rating System or its similar, the Minnesota         Relative Maturity Rating System.     -   MN RM. This represents the Minnesota Relative Maturity Rating         (MN RM) for the hybrid and is based on the harvest moisture of         the grain relative to a standard set of checks of previously         determined MN RM rating. Regression analysis is used to compute         this rating.     -   Yield (Bushels/Acre). The yield is recorded in bushels per acre         of the harvested grain after adjusting to a 15% moisture basis.     -   Moisture. The moisture is the percent grain moisture recorded at         the time of harvest. All hybrids in a test are harvested and         moistures are recorded in succession to insure that there is no         appreciable change in conditions from the recording of the first         hybrid in the test to the recording of the last hybrid in the         test.     -   Growing Degree Units—GDU. Growing degree units (GDU) are heat         units as calculated by the Barger Method. The temperature         maximum and minimums are based on a 24-hour period. Calculations         are from planting. GDU is equal to the maximum temperature plus         the minimum temperature divided by two and subtract 50. If the         maximum temperature is more than 86 degrees then the maximum         temperature is 86. If the minimum temperature is less than 50         degrees then the minimum temperature will be 50. GDU's are a way         of measuring plant maturity.     -   Stalk Lodging. Stalk lodging is a percentage of stalk lodged         plants compared to the total number of plants in a plot. A stalk         lodged plant is defined as a plant that is breaking over at a         point below the upper ear node of attachment.     -   Root Lodging. Root lodging is a percentage of root lodged plants         compared to the total number of plants in a plot. A root lodged         plant is defined as a plant that has the main stalk deviate from         vertical at an approximate angle of 30 degrees or more.     -   Plant Height. This is a measure of the height of the hybrid from         the ground to the tip of the tassel, and is measured in         centimeters.     -   Ear Height. The ear height is a measure from the ground to the         ear node attachment, and is measured in centimeters.     -   Population. The population is a physical count of the number of         plants in a plot and is expressed as the population equivalent         per acre or number of plants per acre.     -   Test Weight. The test weight is recorded in pounds per bushel.         It is a measure of the specific density of the harvested grain.     -   Relative Maturity. Relative maturity is based on the Minnesota         Relative Maturity Rating (MN RM) standard for computing relative         maturity.

DETAILED DESCRIPTION OF THE INVENTION

Inbred corn line KWU7104 is a yellow dent corn with superior characteristics, and provides an excellent parental line in crosses for producing first generation (F₁) hybrid corn. Inbred corn line KWU7104 is best adapted to the Central regions of the United States Corn Belt commonly referred to as Zones 7 and 6. KWU7104 can be used to produce hybrids having a relative maturity of approximately 106 to 113 days on the Comparative Relative Maturity Rating System for harvest moisture of grain. Inbred corn line KWU7104 shows an excellent seedling vigor, an abundant pollen shed, above-average root strength, a superior yield to moisture ratio in the F₁ hybrids when compared to the commonly used Lancaster/KWU7104 based hybrids, and contributes excellent grain fill properties to the F₁ resulting in F₁ ears that fill completely to the tip of the cob. KWU7104 has excellent stay green as an inbred which correlates to the high level of disease tolerance in KWU7104.

KWU7104 is most similar to MO17, however, there are numerous differences including the fact that KWU7104 has a much closer coincidence of the pollen and silk flowering than MO17. Hybrids with KWU7104 are more upright in the leaf attitude and has more girth to the ears.

KWU7104 has a plant height of 191 cm with an average ear insertion of 76 cm. The kernels are arranged in straight rows on the ear. Heat units to 50% pollen shed are approximately 1620 and to 50% silk are approximately 1620.

KWU7104 is an inbred line with very high yield potential in hybrids and an excellent yield to moisture ratio. For an inbred of its maturity, KWU7104 results in unusually large ear size in hybrid combination and the grain fills completely to the tip of the cob. Often these hybrid combinations results in plants which are of much better than average in the root strength when compared to inbred lines of similar maturity.

Some of the criteria used to select ears in various generations include: yield, stalk quality, root quality, disease tolerance, late plant greenness, late season plant intactness, ear retention, ear height, pollen shedding ability, silking ability, and corn borer tolerance. During the development of the line, crosses were made to inbred testers for the purpose of estimating the line's general and specific combining ability, and evaluations were run by the Champaign, Ill. Research Station. The inbred was evaluated further as a line and in numerous crosses by the Champaign station and other research stations across the Corn Belt. The inbred has proven to have a good combining ability in hybrid combinations.

The inbred line has shown uniformity and stability for the traits, as described in the following variety description information. It has been self-pollinated a sufficient number of generations with careful attention to uniformity of plant type. The line has been increased with continued observation for uniformity. No variant traits have been observed or are expected in KWU7104. Inbred corn line, KWU7104 has the following morphological characteristics and is compared to the morphological characteristics of a commonly known inbred, Mo17.

Inbred Description Information

All colors follow the Munsell Color Code. Angles are recorded in degrees. Linear measurements and weights are recorded in the appropriate metric units. TABLE 1 Type: Dent Region Where Developed: Champaign, Illinois GDU 50% Pollen 67 days 1620 heat units GDU 50% Silk 67 1620 heat units Growing Degree Units=GDU=Heat Units as calculated by the Barger Method. The temperature maximum and minimums are based on a 24-hour period. Calculations are from Planting. GDU=((Max Temp+Min Temp)/2)-50

If Max Temp is >86 degrees, then Max Temp=86

If Min Temp is <50 degrees, then Min Temp=50 PLANT Plant Height, Soil to Tassel Tip  191 cm Ear Height, Upper Ear Attachment   76 cm Average Length of 1^(st) Full Internode 12.0 cm Number of Tillers 0 Anthocyanin of Brace Roots Absent

LEAF Width of Ear Node Leaf  7 cm Length of Ear Node Leaf 66 cm No. of Leaves Above Top Ear  5 Leaf Angle from 2^(nd) Leaf above ear at anthesis to Stalk 60° above leaf: Leaf Color  5GY 7/8 Leaf Sheath Pubescense (1 = None, 9 = Like Peach Fuzz):  6 Marginal Waves on Leaf (1 = None, 9 = Many):  6 Longitudinal Leaf Creases (1 = None, 9 = Many):  2

TASSEL Number of Lateral Branches 8 Branch Angle from Central Spike: 20° Tassel Length (Top Leaf Collar to Tassel Tip): 35 cm Pollen Shed (0 = Male Sterile, 9 = Heavy Shed): 8 Anther Color 10Y 9/8 Glume Color 5GY 6/6

EAR: (UNHUSKED) Silk Color 3 Days after Emergence: 10RP ⅝ Fresh Husk Color, 25 Days after 50% Silking: 5GY 6/8 Dry Husk Color, 65 Days after 50% Silking: 5GY 8.5/4 Position of Ear Upright Husk Tightness (1 = Very Loose, 9 = Very Tight): 5 Husk Extension at Harvest Medium

EAR: (HUSKED) Ear Length 16.3 cm Ear Diameter at Mid-Point   41 mm Ear Weight 115.3 grams Number of Kernel Rows, Mid-Ear 14-16 Kernel Rows Alignment Straight Shank Length 10.0 cm Ear Taper Very slight taper

KERNEL: (DRIED) Kernel Length 10.5 mm Kernel Width  8.1 mm Kernel Thickness  5.8 mm Round Kernels, (Shape Grade) 93% Aleurone Color Pattern Homozygous Aleurone Color: White Hard Endosperm Color 10YR 7/10 Endosperm Type: Dent Weight per 100 Kernels   34 gm

COB Cob Diameter at Mid-Point 25 mm Cob Color Red

Agronomic Traits

-   -   Stay Green (@65 days after anthesis) (1-worst to 9-excellent): 8     -   0% Dropped Ears (at 65 days after anthesis)     -   0% Pre-anthesis Brittle Snapping     -   0% Pre-anthesis Root Lodging     -   0% Post-anthesis Root Lodging (at 65 days after anthesis)     -   Yield of inbred per se (at 12-13% grain moisture): 47         bushels/acre

Tables

In Tables 2-4 that follows, the traits and characteristics of inbred corn KWU7104 are given in hybrid combination. The data collected on inbred corn line KWU7104 is presented for the key characteristics and traits. The tables present yield test information about KWU7104. KWU7104 was tested in several hybrid combinations at numerous locations, with two or three replications per location. Information about these hybrids, as compared to several check hybrids, is presented.

The first pedigree listed in the comparison group is the hybrid containing KWU7104. Information for the pedigree includes:

-   -   1. Mean yield of the hybrid across all locations (bu/ac) is         shown in column 2.     -   2. A mean for the percentage moisture (% Moist) for the hybrid         across all locations is shown in column 3.     -   3. A mean of the yield divided by the percentage moisture (Y/M)         for the hybrid across all locations is shown in column 4.     -   4. A mean of the percentage of plants with stalk lodging (%         Stalk) across all locations is shown in column 5.     -   5. A mean of the percentage of plants with root lodging (% Root)         across all locations is shown in column 6.     -   6. Test weight is the grain density measured in pounds per         bushel is shown in column 7.     -   7. Stay Green is a rating made by a trained person as the         hybrids approach maturity. A scale of 1 =lowest to 9         =highest/most desirable is used and is listed in column 8.     -   8. Harvest Appearance is a rating made by a trained person on         the date of harvest. Harvest appearance is the raters impression         of the hybrid based on, but not limited to, a combination of         factors that include plant intactness, tissue health appearance         and ease of harvest as it relates to stalk lodging and root         lodging. A scale of 1 =lowest and 9 =highest/most desirable is         used and is listed in column 9.     -   9. Population is the actual counted plants per harvested plot         area calculated on a plants per acre basis and is shown in         column 10. Figures are given in thousands).     -   10. Plant height is a physical measurement taken from the ground         level to the tip of the tassel. It is expressed to the nearest         inch and is shown in column 11.

11. Ear height is a physical measurement taken from the ground level to the node of attachment for the upper ear. It is expressed to the nearest inch and is shown in column 12. TABLE 2 Overall Comparisons KW4773 × KWU7104 Hybrid versus Check Hybrids - 4 Row Plots Locations: 2002 Data from 27 Testing Locations (NE-3, IA-3, IL-8, IN-8, OH-2, KY-3) % % % Stay Hvst Plt Ear Pedigree Bu/ac Moist Y/M Stalk Root TWT Green App. Pop Ht Ht KW4773 × KWU7104 170.9 18.0 9.5 8.8 4.6 55.5 5.8 5.1 28 104 39 P33P67 168.8 19.8 8.5 5.6 0.7 57.1 5.9 5.3 27 114 42 KW4773 × LH287 163.4 18.4 8.9 12.5 11.6 53.6 6.6 5.5 28 111 40 HC33 × LH287 163.0 16.7 9.8 4.6 2.4 55.0 4.8 5.6 28 110 35 FR1064 × FR4341 160.7 19.1 8.4 4.7 1.6 54.4 5.4 4.9 29 116 48 P31G98 155.6 19.3 8.1 4.3 2.9 55.5 5.9 5.6 28 110 37 MBS3705 × LH287 149.6 19.2 7.8 4.0 1.6 56.2 5.3 4.6 28 110 37 LH310 × LH287 145.9 19.7 7.4 4.9 4.5 52.9 5.9 5.5 27 116 44 LH244 × TR7322 145.3 17.8 8.2 4.3 0.3 54.9 5.8 4.9 28 114 46 LH244 × LH351 144.4 19.3 7.5 6.8 7.4 54.8 5.5 5.1 27 116 46

TABLE 3 Overall Comparisons KW4635 × KWU7104 Hybrid versus Check Hybrids - 4 Row Plots Locations: 2001 Data from 9 Testing Locations (NE-2, IL-4, IN-2, OH-1) % % % Stay Hvst Plt Ear Pedigree Bu/ac Moist Y/M Stalk Root TWT Green App. Pop Ht Ht KW4635 × KWU7104 195.6 20.2 9.7 3.7 0.3 54.0 6.5 6.6 28 108 44 P33G26 190.9 19.0 10.0 6.3 0.6 57.5 5.0 5.6 30 119 46 P33P67 190.6 21.5 8.9 6.5 3.4 56.1 3.0 6.2 29 117 45 KW4635 × KW7638 189.4 20.8 9.1 6.7 0.4 54.9 8.0 7.0 31 106 38 P31G98 185.4 21.4 8.7 6.9 0.5 54.2 5.5 6.6 31 119 55 HC33 × LH283 173.3 18.3 9.5 6.3 3.1 56.4 4.0 4.8 31 113 42 LH246 × KW7638 173.0 21.5 8.0 4.3 0.0 55.9 5.5 6.6 29 107 44

TABLE 4 Overall Comparisons KW4773 × KWU7104 Hybrid versus Check Hybrids - 4 Row Plots Locations: 2001 Data from 14 Testing Locations (NE-2, IL-5, IN-5, OH-1, KY-1) % % % Stay Hvst Plt Ear Pedigree Bu/ac Moist Y/M Stalk Root TWT Green App. Pop Ht Ht KW4773 × KWU7104 184.2 19.4 9.5 6.0 4.6 54.7 6.5 6.7 27 109 44 KW4773 × LH253 184.1 20.3 9.1 5.7 6.5 52.9 7.0 5.8 27 124 51 KW4635 × KW7751 183.7 20.6 8.9 5.6 5.3 54.1 7.3 6.3 28 114 37 KW4635 × KW7638 183.6 20.1 9.1 5.8 5.5 55.5 73.0 6.6 30 112 37 KW4635 × TR7322 180.0 19.0 9.5 6.0 4.1 54.4 5.3 6.4 27 117 46 P33G26 178.1 18.1 9.8 6.2 8.5 57.1 4.0 5.0 29 124 52 HC33 × LH283 177.6 18.1 9.8 6.2 11.4 56.2 5.5 5.1 30 113 40 KW4773 × FR4901 176.4 20.2 8.7 5.5 3.5 54.2 8.0 7.0 29 121 43 KW4635 × FR4901 170.4 20.0 8.5 5.4 3.5 54.0 8.0 6.7 28 119 48

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a corn plant by crossing a first parent corn plant with a second parent corn plant wherein either the first or second parent corn plant is an inbred corn plant of the line KWU7104. Further, both first and second parent corn plants can come from the inbred corn line KWU7104. Still further, this invention also is directed to methods for producing an inbred corn line KWU7104-derived corn plant by crossing inbred corn line KWU7104 with a second corn plant and growing the progeny seed, and repeating the crossing and growing steps with the inbred corn line KWU7104-derived plant from 0 to 7 times. Thus, any such methods using the inbred corn line KWU7104 are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using inbred corn line KWU7104 as a parent are within the scope of this invention, including plants derived from inbred corn line KWU7104. Advantageously, the inbred corn line is used in crosses with other, different, corn inbreds to produce first generation (F₁) corn hybrid seeds and plants with superior characteristics.

It should be understood that the inbred can, through routine manipulation of cytoplasmic or other factors, be produced in a male-sterile form. Such embodiments are also contemplated within the scope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which corn plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, flowers, kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers, silk and the like.

Duncan, et al., Planta 165:322-332 (1985) reflects that 97% of the plants cultured that produced callus were capable of plant regeneration. Subsequent experiments with both inbreds and hybrids produced 91% regenerable callus that produced plants. In a further study in 1988, Songstad, et al., Plant Cell Reports 7:262-265 (1988), reports several media additions that enhance regenerability of callus of two inbred lines. Other published reports also indicated that “nontraditional” tissues are capable of producing somatic embryogenesis and plant regeneration. K. P. Rao et al., Maize Genetics Cooperation Newsletter, 60:64-65 (1986), refers to somatic embryogenesis from glume callus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987) indicates somatic embryogenesis from the tissue cultures of corn leaf segments. Thus, it is clear from the literature that the state of the art is such that these methods of obtaining plants are, and were, “conventional” in the sense that they are routinely used and have a very high rate of success.

Tissue culture of corn is described in European Patent Application, publication 160,390, incorporated herein by reference. Corn tissue culture procedures are also described in Green and Rhodes, “Plant Regeneration in Tissue Culture of Maize,” Maize for Biological Research (Plant Molecular Biology Association, Charlottesville, Va. 367-372, (1982)) and in Duncan et al., “The Production of Callus Capable of Plant Regeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165 Planta 322:332 (1985). Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce corn plants having the physiological and morphological characteristics of inbred corn line KWU7104.

The utility of inbred corn line KWU7104 also extends to crosses with other species. Commonly, suitable species will be of the family Graminaceae, and especially of the genera Zea, Tripsacum, Croix, Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae. Potentially suitable for crosses with KWU7104 may be the various varieties of grain sorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genome of plants to contain and express foreign genes, or additional, or modified versions of native, or endogenous, genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Such foreign additional and/or modified genes are referred to herein collectively as Atransgenes. Over the last fifteen to twenty years several methods for producing transgenic plants have been developed, and the present invention, in particular embodiments, also relates to transformed versions of the claimed inbred line.

Plant transformation involves the construction of an expression vector which will function in plant cells. Such a vector comprises DNA comprising a gene under control of or operatively linked to a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed corn plants, using transformation methods as described below to incorporate transgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation

Marker Genes—Expression vectors include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from transposon Tn5, which when placed under the control of plant regulatory signals confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant Mol Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or broxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643 (1990).

Another class of marker genes for plant transformation require screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS, β-galactosidase, luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3:1681 (1984). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

Recently, in vivo methods for visualizing GUS activity that do not require destruction of plant tissue have been made available. Molecular Probes publication 2908, Imagene GreenJ, p. 1-4 (1993) and Naleway et al., J. Cell Biol. 115:151a (1991). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds and limitations associated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFP may be used as screenable markers.

Promoters—Genes included in expression vectors must be driven by nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are now well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.

As used herein, A promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A plant promoter is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as A tissue preferred. Promoters which initiate transcription only in certain tissue are referred to as tissue specific. A cell type specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An inducible promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of non-constitutive promoters. A constitutive promoter is a promoter which is active under most environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression in corn. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in corn. With an inducible promoter the rate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression in corn or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in corn.

Many different constitutive promoters can be utilized in the instant invention. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985) and the promoters from such genes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); PEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xba1/Ncol fragment), represents a particularly useful constitutive promoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expression in corn. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in corn. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondroin or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5′ and/or 3′ region of a gene encoding the protein of interest. Targeting sequences at the 5′ and/or 3′ end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S., Master's Thesis, Iowa State University (1993), Knox, C., et al., AStructure and Organization of Two Divergent Alpha-Amylase Genes from Barley, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, et al., A short amino acid sequence able to specify nuclear location, Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants which are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods which are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided for commercial production of foreign protein is corn. In another preferred embodiment, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton 269:284 (1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants, to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

-   -   A. Plant disease resistance genes. Plant defenses are often         activated by specific interaction between the product of a         disease resistance gene (R) in the plant and the product of a         corresponding avirulence (Avr) gene in the pathogen. A plant         inbred line can be transformed with cloned resistance gene to         engineer plants that are resistant to specific pathogen strains.         See, for example Jones et al., Science 266:789 (1994) (cloning         of the tomato Cf-9 gene for resistance to Cladosporium fulvum);         Martin et al., Science 262:1432 (1993) (tomato Pto gene for         resistance to Pseudomonas syringae pv. Tomato encodes a protein         kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2         gene for resistance to Pseudomonas syringae).     -   B. A Bacillus thuringiensis protein, a derivative thereof or a         synthetic polypeptide modeled thereon. See, for example, Geiser         et al., Gene 48:109 (1986), who disclose the cloning and         nucleotide sequence of a Bt δ-endotoxin gene. Moreover, DNA         molecules encoding δ-endotoxin genes can be purchased from         American Type Culture Collection, Manassas, Va., for example,         under ATCC Accession Nos. 40098, 67136, 31995 and 31998.     -   C. A lectin. See, for example, the disclose by Van Damme et al.,         Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide         sequences of several Clivia miniata mannose-binding lectin         genes.     -   D. A vitamin-binding protein such as avidin. See PCT application         US93/06487, the contents of which are hereby incorporated by         reference. The application teaches the use of avidin and avidin         homologues as larvicides against insect pests.     -   E. An enzyme inhibitor, for example, a protease or proteinase         inhibitor or an amylase inhibitor. See, for example, Abe et         al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of         rice cysteine proteinase inhibitor), Huub et al., Plant Molec.         Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding         tobacco proteinase inhibitor I), Sumitani et al., Biosci.         Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of         Streptomyces nitrosporeus α-amylase inhibitor).     -   F. An insect-specific hormone or pheromone such as an         ecdysteroid and juvenile hormone, a variant thereof, a mimetic         based thereon, or an antagonist or agonist thereof. See, for         example, the disclosure by Hammock et al., Nature 344:458         (1990), of baculovirus expression of cloned juvenile hormone         esterase, an inactivator of juvenile hormone.     -   G. An insect-specific peptide or neuropeptide which, upon         expression, disrupts the physiology of the affected pest. For         example, see the disclosures of Regan, J. Biol. Chem.         269:9 (1994) (expression cloning yields DNA coding for insect         diuretic hormone receptor), and Pratt et al., Biochem. Biophys.         Res. Comm. 163:1243 (1989) (an allostatin is identified in         Diploptera puntata). See also U.S. Pat. No. 5,266,317 to         Tomalski et al., who disclose genes encoding insect-specific,         paralytic neurotoxins.     -   H. An insect-specific venom produced in nature by a snake, a         wasp, etc. For example, see Pang et al., Gene 116:165 (1992),         for disclosure of heterologous expression in plants of a gene         coding for a scorpion insectotoxic peptide.     -   I. An enzyme responsible for a hyper accumulation of a         monterpene, a sesquiterpene, a steroid, hydroxamic acid, a         phenylpropanoid derivative or another non-protein molecule with         insecticidal activity.     -   J. An enzyme involved in the modification, including the         post-translational modification, of a biologically active         molecule; for example, a glycolytic enzyme, a proteolytic         enzyme, a lipolytic enzyme, a nuclease, a cyclase, a         transaminase, an esterase, a hydrolase, a phosphatase, a kinase,         a phosphorylase, a polymerase, an elastase, a chitinase and a         glucanase, whether natural or synthetic. See PCT application WO         93/02197 in the name of Scott et al., which discloses the         nucleotide sequence of a callase gene. DNA molecules which         contain chitinase-encoding sequences can be obtained, for         example, from the ATCC under Accession Nos. 39637 and 67152. See         also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993),         who teach the nucleotide sequence of a cDNA encoding tobacco         hookworm chitinase, and Kawalleck et al., Plant Molec. Biol.         21:673 (1993), who provide the nucleotide sequence of the         parsley ubi4-2 polyubiquitin gene.     -   K. A molecule that stimulates signal transduction. For example,         see the disclosure by Botella et al., Plant Molec. Biol. 24:757         (1994), of nucleotide sequences for mung bean calmodulin cDNA         clones, and Griess et al., Plant Physiol. 104:1467 (1994), who         provide the nucleotide sequence of a maize calmodulin cDNA         clone.     -   L. A hydrophobic moment peptide. See PCT application WO95/16776         (disclosure of peptide derivatives of Tachyplesin which inhibit         fungal plant pathogens) and PCT application WO95/18855 (teaches         synthetic antimicrobial peptides that confer disease         resistance), the respective contents of which are hereby         incorporated by reference.     -   M. A membrane permease, a channel former or a channel blocker.         For example, see the disclosure of Jaynes et al., Plant Sci         89:43 (1993), of heterologous expression of a cecropin-β, lytic         peptide analog to render transgenic tobacco plants resistant to         Pseudomonas solanacearum.     -   N. A viral-invasive protein or a complex toxin derived         therefrom. For example, the accumulation of viral coat proteins         in transformed plant cells imparts resistance to viral infection         and/or disease development effected by the virus from which the         coat protein gene is derived, as well as by related viruses. See         Beachy et al., Ann. rev. Phytopathol. 28:451 (1990). Coat         protein-mediated resistance has been conferred upon transformed         plants against alfalfa mosaic virus, cucumber mosaic virus,         tobacco streak virus, potato virus X, potato virus Y, tobacco         etch virus, tobacco rattle virus and tobacco mosaic virus. Id.     -   O. An insect-specific antibody or an immunotoxin derived         therefrom. Thus, an antibody targeted to a critical metabolic         function in the insect gut would inactivate an affected enzyme,         killing the insect. Cf. Taylor et al., Abstract #497, Seventh         Int'l Symposium on Molecular Plant-Microbe Interactions         (Edinburgh, Scotland) (1994) (enzymatic inactivation in         transgenic tobacco via production of single-chain antibody         fragments).     -   P. A virus-specific antibody. See, for example, Tavladoraki et         al., Nature 366:469 (1993), who show that transgenic plants         expressing recombinant antibody genes are protected from virus         attack.     -   Q. A developmental-arrestive protein produced in nature by a         pathogen or a parasite. Thus, fungal endo         α-1,4-D-polygalacturonases facilitate fungal colonization and         plant nutrient release by solubilizing plant cell wall         homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology         10:1436 (1992). The cloning and characterization of a gene which         encodes a bean endopolygalacturonase-inhibiting protein is         described by Toubart et al., Plant J. 2:367 (1992).     -   R. A development-arrestive protein produced in nature by a         plant. For example, Logemann et al., Bioi/Technology 10:305         (1992), have shown that transgenic plants expressing the barley         ribosome-inactivating gene have an increased resistance to         fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example:

-   -   A. A herbicide that inhibits the growing point or meristem, such         as an imidazalinone or a sulfonylurea. Exemplary genes in this         category code for mutant ALS and AHAS enzyme as described, for         example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al.,         Theor. Appl. Genet. 80:449 (1990), respectively.     -   B. Glyphosate (resistance impaired by mutant         5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,         respectively) and other phosphono compounds such as glufosinate         (phosphinothricin acetyl transferase, PAT and Streptomyces         hygroscopicus phosphinothricin-acetyl transferase, bar, genes),         and pyridinoxy or phenoxy propionic acids and cycloshexones         (ACCase inhibitor-encoding genes). See, for example, U.S. Pat.         No. 4,940,835 to Shah, et al., which discloses the nucleotide         sequence of a form of EPSP which can confer glyphosate         resistance. A DNA molecule encoding a mutant aroA gene can be         obtained under ATCC accession number 39256, and the nucleotide         sequence of the mutant gene is disclosed in U.S. Pat. No.         4,769,061 to Comai. European patent application No. 0 333 033 to         Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al.,         disclose nucleotide sequences of glutamine synthetase genes         which confer resistance to herbicides such as         L-phosphinothricin. The nucleotide sequence of a         phosphinothricin-acetyl-transferase gene is provided in European         application No. 0 242 246 to Leemans et al., DeGreef et al.,         Bio/Technology 7:61 (1989), describe the production of         transgenic plants that express chimeric bar genes coding for         phosphinothricin acetyl transferase activity. Exemplary of genes         conferring resistance to phenoxy propionic acids and         cycloshexones, such as sethoxydim and haloxyfop are the Acc1-S1,         Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theor.         Appl. Genet. 83:435 (1992).     -   C. A herbicide that inhibits photosynthesis, such as a triazine         (psbA and gs+ genes) and a benzonitrile (nitrilase gene).         Przibilla et al., Plant Cell 3:169 (1991), describe the         transformation of Chlamydomonas with plasmids encoding mutant         psbA genes. Nucleotide sequences for nitrilase genes are         disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA         molecules containing these genes are available under ATCC         Accession Nos. 53435, 67441, and 67442. Cloning and expression         of DNA coding for a glutathione S-transferase is described by         Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, such as:

-   -   A. Modified fatty acid metabolism, for example, by transforming         a plant with an antisense gene of stearyl-ACP desaturase to         increase stearic acid content of the plant. See Knultzon et al.,         Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992).     -   B. Decreased phytate content     -   1) Introduction of a phytase-encoding gene would enhance         breakdown of phytate, adding more free phosphate to the         transformed plant. For example, see Van Hartingsveldt et al.,         Gene 127:87 (1993), for a disclosure of the nucleotide sequence         of an Aspergillus niger phytase gene.     -   2) A gene could be introduced that reduced phytate content. In         maize, this, for example, could be accomplished, by cloning and         then reintroducing DNA associated with the single allele which         is responsible for maize mutants characterized by low levels of         phytic acid. See Raboy et al., Maydica 35:383 (1990).     -   C. Modified carbohydrate composition effected, for example, by         transforming plants with a gene coding for an enzyme that alters         the branching pattern of starch. See Shiroza et al., J. Bacteol.         170:810 (1988) (nucleotide sequence of Streptococcus mutants         fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.         20:220 (1985) (nucleotide sequence of Bacillus subtilis         levansucrase gene), Pen et al., Bio/Technology 10:292 (1992)         (production of transgenic plants that express Bacillus         lichenifonnis α-amylase), Elliot et al., Plant Molec. Biol.         21:515 (1993) (nucleotide sequences of tomato invertase genes),         Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directed         mutagenesis of barley a-amylase gene), and Fisher et al., Plant         Physiol. 102:1045 (1993) (maize endosperm starch branching         enzyme II).         Methods for Corn Transformation

Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic, soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediated transformation is broad, some major cereal crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992). In corn, several target tissues can be bombarded with DNA-coated microprojectiles in order to produce transgenic plants, including, for example, callus (Type I or Type II), immature embryos, and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol or poly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994).

Following transformation of corn target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used for producing a transgenic inbred line. The transgenic inbred line could then be crossed, with another (non-transformed or transformed) inbred line, in order to produce a new transgenic inbred line. Alternatively, a genetic trait which has been engineered into a particular corn line using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred line into an elite inbred line, or from an inbred line containing a foreign gene in its genome into an inbred line or lines which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the present invention, this also includes any single gene conversions of that inbred. The term single gene converted plant as used herein refers to those corn plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of an inbred are recovered in addition to the single gene transferred into the inbred via the backcrossing technique. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the inbred. The term backcrossing as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental corn plants for that inbred. The parental corn plant which contributes the gene for the desired characteristic is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental corn plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the original inbred of interest (recurrent parent) is crossed to a second inbred (nonrecurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a corn plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original inbred. To accomplish this, a single gene of the recurrent inbred is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological, constitution of the original inbred. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross, one of the major purposes is to add some commercially desirable, agronomically important trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularly selected for in the development of a new inbred but that can be improved by backcrossing techniques. Single gene traits may or may not be transgenic, examples of these traits include but are not limited to, male sterility, waxy starch, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, male fertility, enhanced nutritional quality, industrial usage, yield stability and yield enhancement. These genes are generally inherited through the nucleus. Some known exceptions to this are the genes for male sterility, some of which are inherited cytoplasmically, but still act as single gene traits. Several of these single gene traits are described in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of which are specifically hereby incorporated by reference.

Industrial Applicability

Corn is used as human food, livestock feed, and as raw material in industry. The food uses of corn, in addition to human consumption of corn kernels, include both products of dry- and wet-milling industries. The principal products of corn dry milling are grits, meal and flour. The corn wet-milling industry can provide corn starch, corn syrups, and dextrose for food use. Corn oil is recovered from corn germ, which is a by-product of both dry- and wet-milling industries.

Corn, including both grain and non-grain portions of the plant, is also used extensively as livestock feed, primarily for beef cattle, dairy cattle, hogs and poultry.

Industrial uses of corn include production of ethanol, corn starch in the wet-milling industry and corn flour in the dry-milling industry. The industrial applications of corn starch and flour are based on functional properties, such as viscosity, film formation, adhesive properties, and ability to suspend particles. The corn starch and flour have application in the paper and textile industries. Other industrial uses include applications in adhesives, building materials, foundry binders, laundry starches, explosives, oil-well muds and other mining applications.

Plant parts other than the grain of corn are also used in industry, for example: stalks and husks are made into paper and wallboard and cobs are used for fuel and to make charcoal.

The seed of inbred corn line KWU7104, the plant produced from the inbred seed, the hybrid corn plant produced from the crossing of the inbred, hybrid seed, and various parts of the hybrid corn plant and transgenic versions of the foregoing, can be utilized for human food, livestock feed, and as a raw material in industry.

Deposit Information

A deposit of seed of the corn inbred, KWU7104, of this invention is maintained by AgReliant Genetics, LLC 972 County Road 500 East, Ivesdale, Ill. 61851. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patent and Trademarks to be entitled thereto under 37 CRF 1.14 and 35 USC 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the inbred will be irrevocably removed by affording access to a deposit of at least 2,500 seeds of the same inbred with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110

Although the forgoing invention has been described in some detail by way of illustration and example for purpose of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims. 

1. Seed of corn inbred line designated KWU7104, representative seed of said line having been deposited under ATCC Accession No. PTA-______.
 2. A corn plant, or a part thereof, produced by growing the seed of claim
 1. 3. The corn plant of claim 2, wherein said plant has been detasseled.
 4. A tissue culture of regenerable cells produced from the plant of claim
 2. 5. Protoplasts produced from the tissue culture of claim
 4. 6. The tissue culture of claim 4, wherein cells of the tissue culture are from a tissue selected from the group consisting of leaf, pollen, embryo, root, root tip, anther, silk, flower, kernel, ear, cob, husk and stalk.
 7. A corn plant regenerated from the tissue culture of claim 4, said plant having all the morphological and physiological characteristics of inbred line KWU7104, representative seed of said line having been deposited under ATCC Accession No. PTA-______.
 8. A method for producing an F1 hybrid corn seed, comprising crossing the plant of claim 2 with a different corn plant and harvesting the resultant F1 hybrid corn seed.
 9. A hybrid corn seed produced by the method of claim
 8. 10. A hybrid corn plant, or parts thereof, produced by growing said hybrid seed of claim
 9. 11. A method for producing a male sterile corn plant comprising transforming the corn plant of claim 2 with a nucleic acid molecule that confers male sterility.
 12. A male sterile corn plant produced by the method of claim
 11. 13. A method of producing an herbicide resistant corn plant comprising transforming the corn plant of claim 2 with a transgene that confers herbicide resistance.
 14. An herbicide resistant corn plant produced by the method of claim
 13. 15. The corn plant of claim 14, wherein the transgene confers resistance to an herbicide selected from the group consisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.
 16. A method of producing an insect resistant corn plant comprising transforming the corn plant of claim 2 with a transgene that confers insect resistance.
 17. An insect resistant corn plant produced by the method of claim
 16. 18. The corn plant of claim 17, wherein the transgene encodes a Bacillus thuringiensis endotoxin.
 19. A method of producing a disease resistant corn plant comprising transforming the corn plant of claim 2 with a transgene that confers disease resistance.
 20. A disease resistant corn plant produced by the method of claim
 19. 21. A method of producing a corn plant with decreased phytate content comprising transforming the corn plant of claim 2 with a transgene encoding phytase.
 22. A corn plant with decreased phytate content produced by the method of claim
 21. 23. A method of producing a corn plant with modified fatty acid metabolism or modified carbohydrate metabolism comprising transforming the corn plant of claim 2 with a transgene encoding a protein selected from the group consisting of stearyl-ACP desaturase, fructosyltransferase, levansucrase, alpha-amylase, invertase and starch branching enzyme.
 24. A corn plant produced by the method of claim
 23. 25. The corn plant of claim 24 wherein the transgene confers a trait selected from the group consisting of waxy starch and increased amylose starch.
 26. A corn plant, or part thereof, having all the physiological and morphological characteristics of the inbred line KWU7104, representative seed of said line having been deposited under ATCC Accession No. PTA-______.
 27. A method of introducing a desired trait into corn inbred line KWU7104 comprising: (a) crossing KWU7104 plants grown from KWU7104 seed, representative seed of which has been deposited under ATCC Accession No. PTA-______, with plants of another corn line that comprise a desired trait to produce F1 progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, disease resistance and waxy starch; (b) selecting F1 progeny plants that have the desired trait to produce selected F1 progeny plants; (c) crossing the selected progeny plants with the KWU7104 plants to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the desired trait and physiological and morphological characteristics of corn inbred line KWU7104 listed in Table 1 to produce selected backcross progeny plants; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of corn inbred line KWU7104 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 28. A plant produced by the method of claim 27, wherein the plant has the desired trait and all of the physiological and morphological characteristics of corn inbred line KWU7104 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 29. The plant of claim 28 wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.
 30. The plant of claim 28 wherein the desired trait is insect resistance and the insect resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin.
 31. The plant of claim 28 wherein the desired trait is male sterility and the trait is conferred by a cytoplasmic nucleic acid molecule that confers male sterility.
 32. A method of modifying fatty acid metabolism, modified phytic acid metabolism or modified carbohydrate metabolism into corn inbred line KWU7104 comprising: (a) crossing KWU7104 plants grown from KWU7104 seed, representative seed of which has been deposited under ATCC Accession No. PTA-______, with plants of another corn line that comprise a nucleic acid molecule encoding an enzyme selected from the group consisting of phytase, stearyl-ACP desaturase, fructosyltransferase, levansucrase, alpha-amylase, invertase and starch branching enzyme; (b) selecting F1 progeny plants that have said nucleic acid molecule to produce selected F1 progeny plants; (c) crossing the selected progeny plants with the KWU7104 plants to produce backcross progeny plants; (d) selecting for backcross progeny plants that have said nucleic acid molecule and physiological and morphological characteristics of corn inbred line KWU7104 listed in Table 1 to produce selected backcross progeny plants; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise said nucleic acid molecule and have all of the physiological and morphological characteristics of corn inbred line KWU7104 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions.
 33. A plant produced by the method of claim 32, wherein the plant comprises the nucleic acid molecule and has all of the physiological and morphological characteristics of corn inbred line KWU7104 listed in Table 1 as determined at the 5% significance level when grown in the same environmental conditions. 